Conjugates and specific immunoassays for the methadone metabolite 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine

ABSTRACT

Novel chemical analogs of the methadone metabolite 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) are disclosed. The derivatives can be used for formation of EDDP-protein conjugates. The conjugates can be used in turn to raise antibodies reactive with EDDP and having a low cross-reactivity with methadone. The antibodies and EDDP-enzyme polypeptide conjugates provide the basis for specific immunoassays used in monitoring compliance with methadone treatment.

REFERENCE TO RELATED APPLICATIONS

This is a national stage application under 35 USC § 371 of anInternational application filed on the same date. This applicationclaims the priority benefit of U.S. Provisional Application U.S.60/047,773, filed on May 27, 1997.

TECHNICAL FIELD

This invention relates generally to the field of the detection of drugmetabolites in biological samples. More specifically, it provides asystem for developing hapten conjugates and specific antibodies for usein assay systems for detection or quantitation of the title methadonemetabolite.

BACKGROUND ART

Methadone has been widely used as an aid in withdrawal from heroinaddiction. Compliance with methadone therapy is frequently monitored byanalysis of urine samples for the presence of methadone, which can beperformed using one of several commercially available immunoassays formethodone.

However, there are several occasions when a simple assay for methadoneprovides in incorrect or incomplete diagnostic information. For example,the methadone may be so extensively metabolized that the concentrationexcreted falls below that of the assay being used. To the extent thatthe assay distinguishes between methadone and excreted metabolites, thetest can be negative even for a patient in full compliance with therapy(Nicar et al., Clin. Chem. 42:543, 1996). In another example, a patientmay add methadone to their sample to disguise the fact that they are notadhering to the treatment protocol. Samples that have been tampered withcan in principle be distinguished in that they will not containfilterable metabolites of methadone that are also present in urine whenthe methadone treatment protocol is being properly adhered to.

Both these types of situations could be easily recognized if it werepossible to independently measure the presence of the excretedmetabolite. In practical terms, detecting methadone metabolites isdifficult because of the chemical and immunological similarity withmethadone itself. Accordingly, there is a need for reagents andtechniques that would permit routine monitoring of methadone metabolitesin a clinical setting.

A major pathway of metabolism of methadone is demethylation to thepresumed intermediate N-desmethylmethadone, leading to urinary excretionof 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine herein abbreviatedas EDDP), 2-ethyl-5-methyl-3,3-diphenyl-1-pyrroline (EMDP), and theirring-hydroxylated analogs (Sullivan et al., J. Medicinal Chem. 16:909,1973). These structures are depicted in FIG. 1. Lesser amounts of4-dimethylamino-2,2-diphenylvaleric acid (formed by side-chainoxidation); 1 ,5-dimethyl-3,3-diphenyl-2-pyrrolidone (resulting fromsubsequent N-demethylation and cyclization), ring-hydroxylatedmethadone, and normethadol are also found. Methadone N-oxide is formedby storage of samples at 30° C. In human volunteers receiving˜100 mg ofdl-methadone orally, EDDP was found by Sullivan et al. to be the mostprominent metabolite in urine, present at a level roughly comparablewith that of methadone itself.

In order to detect methadone metabolites, Sullivan et al. extractedurine with methylene chloride, hydrolyzed the reconstituted extractusing β-glucuronidase and aryl sulfatase, optionally acetylated ormethylated the product, and then characterized it by gas chromatographyor combined gas chromatography-mass spectroscopy.

The synthesis and spectral properties of optically active EDDP weredescribed by Brine et al. (J. Heterocyclic Chem., 23:369, 1986). The ¹H--NMR analysis suggested that the free base exists predominantly in anenamine form. CD and ORD studies provided data consistent with a fairlyrigid enamine structure.

Simultaneous gas chromatography mass spectrometry (GC/MS) assay for EDDPin urine was described by Baugh et al. (J. Forensic Sci. 36:548, 1991).Urine was extracted with 1-chlorobutane at pH˜9, the organic phase wasback-extracted into acetate buffer, adjusted to pH˜9, and re-extractedwith I -chlorobutane. Area corresponding to ions at m/z 277, 262, and276 was measured. Quantitation was enhanced by using deuteratedmethadone as the internal standard. Although the assay is quantitative,it relies on a multi-step extraction procedure and the availability ofequipment to perform GC/MS.

Thin-layer chromatography (TLC) screening of methadone and EDDP in urinewas described by Budd et al. (Clin. Toxicol. 16:55, 1980). Metaboliteswere extracted into an organic solvent, and then chromatographed inethyl acetate:methanol:diethylamine or ethyl acetate:methylenechoride:propylamine. Dried plates were developed using acidifiediodoplatinate reagent. Although this permits a number of samples to beprocessed in the same day, this type of assay is non-quantitative andsubject to variations in solvent mixtures.

More suited for routine clinical analysis are immunoassays, in which aspecific antibody is used to distinguish and quantify an analyte ofinterest in a biological sample. The general art of immunoassay and itsuse in clinical monitoring is well known. Assays for analytes of thesize of EDDP are often competition assays. Immunoassays typicallyinvolve either the specific isolation of the analyte from the samplemediated by antibody, or the formation of analyte-antibody complexes insitu (a "homogeneous" assay system). In either case, formation of ananalyte-antibody complex ultimately leads to a signal which is directlyor inversely related to the amount of analyte present in the originalsample.

A particularly powerful homogeneous assay system is the cloned enzymedonor immunoassay (CEDIA®), described in U.S. Pat. No. 4,708,929, and inHenderson, Clin. Chem. 32:1637, 1986. In a preferred form of the CEDIA®assay, two subunits of the enzyme β-galactosidase associate to providethe detectable signal, which is quantitatively affected byanalyte-specific antibody except in the presence of a sample containingfree analyte.

All specific immunoassays require the availability of an antibody thatbinds the analyte of interest but not potential interfering substances.An immunoassay for EDDP capable of differentiating samples spiked withmethadone requires an antibody with a very high relative affinity forEDDP in relation to methadone.

A commercial assay for EDDP is marketed by Diagnostic Reagents, Inc. ofMountain View, Calif. This is a homogeneous enzyme immunoassay based onthe binding of an anti-EDDP antibody with a glucose-6-phosphatedehydrogenase drug conjugate, which inhibits the activity of the enzyme.Presence of sample analyte in the reaction mixture binds the antibodyand increases enzyme activity. The utility of this assay is limited byits specificity. EDDP gives a positive result at 300 ng/mL, EMDP at 400ng/mL, and methadone itself at 5000 ng/mL. In other words, the assaywhen used alone is unable to distinguish between a sample containingEDDP, and another sample spiked with methadone at a somewhat higherlevel.

The limited specificity in current art EDDP assays is attributable tothe limited specificity in the antibody in the assay. This in turn isattributable to the unsuitability of current art immunogens forgenerating antibodies with better specificity.

DISCLOSURE OF THE INVENTION

The present invention provides a system for the improved detection ofEDDP in biological samples. New chemical analogs of EDDP are describedthat permit EDDP to be linked to an immunogenic carrier. In turn, theimmunogens can be used reproducibly to generate antibodies with anexquisite ability to distinguish EDDP from methadone, EMDP, and otherpotential interfering substances. The specific antibodies obtained canbe used in any suitable immunoassay format for the detection orquantitation of EDDP in a sample, distinct from any methadone that ispresent. Chemical derivatives of EDDP according to this invention canalso be conjugated to proteins or surfaces as a competitive target forthe antibody in an immunoassay.

Certain embodiments of this invention relate to a purified or syntheticcompound of the formula: ##STR1## and salts thereof, wherein R is asubstituent containing at least one C atom. In certain embodiments, Rcomprises a polyamino acid. In certain embodiments, R is of thestructure Y--R¹ --O--, wherein

R¹ is a hydrocarbyl diradical having 1-10 carbon atoms and

Y is selected from the group consisting of:

--COOH;

--COOR² ; wherein R² is a hydrocarbyl radical having 1-20 carbon atoms;##STR2## wherein R³ is a hydrocarbyl diradical having 1-20 carbon atoms;and --CO--L--Q, wherein Q is a poly(amino acid) and L is a bond or adiradical linker group, such as ##STR3## wherein R⁴ is a hydrocarbyldiradical of 1-20 carbon atoms. Where present, the polyamino acid may bean immunogenic peptide, an enzyme, or an enzyme donor polypeptide.

Additional embodiments of this invention relate to synthetic methods forpreparing the chemical compounds and EDDP-protein conjugates outlinedabove, comprising synthesizing the intermediate2-ethyl-1,5-dimethyl-3-p-hydroxyphenyl-3-phenylpyrroline (p--HO--EDDP)or the intermediate 1,5-dimethyl-3-(p-alkoxyphenyl)-3-phenyl-2-pyrrolidone. Other embodiments relate to methods for preparingEDDP-protein conjugates comprising synthesizing an EDDP derivative ofthis invention with a suitable reactive group for protein conjugation.

Also embodied in this invention are monoclonal and polyclonal antibodiesraised against any of the compounds outlined above and specific for thecompound 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidone (EDDP). Alsoembodied is an antibody specific for EDDP and having a low level ofcross-reactivity with methadone or with the metabolite2-ethyl-5-methyl-3,3-diphenyl-1-pyrroline (EMDP). Also embodied aremethods for raising such antibodies by stimulating an immunocompetentanimal, cell or viral particle with an immunogenic EDDP-proteinconjugate of this invention or using an EDDP-protein conjugate to selecta hybridoma of the desired specificity.

Also embodied in this invention are EDDP-protein conjugates forconducting a competitive assay for EDDP in a sample, wherein the proteinis either coupled to a solid support or capable of generating adetectable signal. The signal may be generated by a radioisotope,fluorochrome or other signal-generating or quenching system attached tothe protein (or by an adaptation that allows the signal-generating orquenching system to be subsequently attached), or the protein may be anenzyme or enzyme component capable of assembling into an active enzymein the presence or absence of EDDP in the sample.

Further embodiments of this invention relate to methods for detectingEDDP in a sample, exemplified by but not limited to a urine sample,comprising combining the sample with the an anti-EDDP antibody of thisinvention under conditions that permit formation of a stableEDDP-antibody complex; and detecting or quantitating any complex formed.Other embodiments relate to methods for detecting EDDP in a sample bycompetition with an EDDP-protein conjugate. Preferred methods aremethods using an enzyme to produce a detectable signal, and homogeneousassay methods such as a cloned enzyme donor immunoassay. Any EDDPdetected in a sample may be correlated with administration of methadoneto a subject, particularly a human on methadone therapy.

Additional embodiments relate to certain reagents embodied in thisinvention, reagent combinations, and kits for performing an assay methodfor EDDP in a biological sample.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a chemical scheme showing urinary metabolites in subjects onmethadone maintenance. Methadone (Structure 1) is metabolized through apresumed intermediate to the major metabolite EDDP (Structure 2) and theminor metabolite EMDP (Structure 3), which may be ring-hydroxylated toStructures 6 and 5, respectively. This scheme is adapted from Sullivanet al. (supra).

FIG. 2 is a drawing of an EDDP analog designated EMDP--MPS. This analogwas obtained by derivatizing the N in the heterocyclic ring of EMDP.Antibodies raised using EDDP-protein conjugates based on this analoggenerally had an unsatisfactory degree of cross-reactivity withmethadone.

FIG. 3 is a chemical scheme showing the synthesis of4-dimethylamino-2-(p-alkoxyphenyl)-2-phenylpentane-nitrile, where thealkoxyphenyl group is methoxyphenyl.

FIG. 4 is a chemical scheme showing the synthesis of a1,5-dimethyl-3-(p-alkoxyphenyl)-3-phenyl-2-pyrrolidone, where thealkoxyphenyl group is methoxyphenyl.

FIG. 5 is a chemical scheme showing the synthesis of the keyintermediate 2-ethyl-1,5-dimethyl-3-p-hydroxyphenyl-3-phenylpyrroline(p--HO--EDDP) as a hydrochloride salt. Availability of p--HO--EDDP makespossible the preparation of a new family of EDDP chemical analogs andEDDP-protein conjugates as described in this disclosure.

FIG. 6 is a drawing of two diastereomers of p--HO--EDDP.

FIG. 7 is a chemical scheme showing the synthesis of thet-butyl-carboxymethyl ether of EDDP (p--t--Bu--CME--EDDP).

FIG. 8 is a chemical scheme showing the deprotection ofp--t--Bu--CME--EDDP to yield the carboxymethyl ether of p--HO--EDDP(p--CME--EDDP). p--CME--EDDP and other carboxylated analogs of EDDP canbe condensed with free amino groups on proteins to form EDDP-proteinconjugates linked through an amide bond.

FIG. 9 is a chemical scheme showing the coupling of p--CME--EDDP withmaleimidoethylamine (MEA) to yield the maleimide adductp--MEA--CME--EDDP. This and other maleimide adducts of EDDP can belinked to proteins via thiol groups present as free cysteine side-chainsor by thiolation of the protein with a reagent such as 2-iminothiolane(IT).

FIG. 10 is a line graph showing a cloned enzyme donor immunoassay EDDPimmunoassay calibration curve, using the EDDP-specific monoclonalantibodies designated 14C4 (Panel A) and 14G4 (Panel B).

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention is focused on the preparation of analogs of themethadone metabolite EDDP, which can then be used for preparingimmunogens and enzyme conjugates useful in the development ofimmunoassays for the measurement of EDDP.

Upon facing the challenge of developing EDDP conjugates, a typicalsynthetic approach is to derivatize from the N residue in thepyrrolidone ring. EMDP, which lacks the N-methyl group of EDDP, iscommercially available and a suitable starting compound. The C=N doublebond is reduced to yield the secondary amine, which can then be attachedto a linking group.

This approach was adopted in a preliminary series of experiments. TheEDDP analog EMDP--MPS with the structure shown in FIG. 2 was synthesizedand conjugated to KLH that has been thiolated using 2-iminothiolane. Thehapten-protein conjugate was used as an immunogen to prepare monoclonalantibodies. Five separate fusions were performed using immunocytes fromhyperimmunized mice. The monoclonal antibodies obtained had across-reactivity with methadone of 0.8 to 5.6% and a cross-reactivitywith EMDP of 1.0-6.8%.

With a view to obtaining antibodies with improved ability todiscriminate between these structures, an entirely different syntheticapproach was developed. A novel family of EDDP analogs was obtained byconjugating from the para position of a phenyl group of EDDP. Toaccomplish this, a synthetic method was developed for the preparation ofpara-hydroxylated EDDP,1,5-Dimethyl-3-(p-hydroxyphenyl)-3-phenyl-2-pyrrolidone (abbreviatedherein as p--HO--EDDP). This structure has previously been identified asyet another methadone metabolite, but has not previously beensynthesized. p--HO--EDDP was then alkylated to yield a carboxymethylether (p--CME--EDDP). This was used to prepare protein conjugates eitherby direct condensation or via the linking group maleimidoethylamine(p--MEA--CME--EDDP).

It was found that p--HO--EDDP derivatives are much more effective forgenerating specific anti-EDDP antibodies than prior art compounds. Whencoupled to the protein carrier KLH, both p--CME--EDDP andp--MEA--CME--EDDP elicited a number of specific antibody clones withminimal cross-reactivity to methadone or EMDP.

The synthesis of the EDDP analogs of this invention is accomplished byfirst preparing the intermediate, para-hydroxy-EDDP (p--HO--EDDP). Thesynthesis is accomplished as follows: The starting materialp-methoxybenzophenone is commercially available. This is elaborated togive the nitrile shown in FIG. 3. The nitrile is converted into the1,5-dimethyl-3-p-methoxyphenyl-3-phenyl-2-pyrrolidone using the reactionscheme shown in FIG. 4.

In order to complete the synthesis, a more acid labile HO-protectinggroup than methoxy is used to prevent decomposition of the EDDP enamineduring the final steps. A protecting group is chosen that is stable tothe strongly basic conditions used when converting the pyrrolidone tothe 2-ethylidene function by reacting with ethyllithium. Use of t-butylether as a protecting group fulfills these requirements. Thus, thep-methoxy-pyrrolidone is converted to the p-hydroxy-pyrrolidone bytreatment with boron tribromide. The hydroxy group is converted into at-butyl ether by acid catalyzed reaction with isobutylene to give thep--t-butoxy-pyrrolidone. This intermediate is reacted with ethyllithiumto give the p--t-butoxy-EDDP. Finally, treatment with HCl in dioxanegives the p--HO--EDDP as a hydrochloride salt (FIG. 5).

The product of this procedure is a mixture of diastereomers as shown inFIG. 6,, which may be confirmed by HPLC or NMR.

The artisan of ordinary skill in the art will appreciate that theavailability of preparative amounts of pure or synthetic p--HO--EDDPprovides a gateway to a number of EDDP analogs, including those of thegeneral form: ##STR4##

For example, the phenol group can be modified or conjugated according toany one of a number of standard chemical synthetic procedures, such asether and ester formation, ring substitution and the like. Preferredembodiments are those in which R is a substituent having one or more Nor C atoms, particularly an ether, ester, thioester, amine, or amide,and derivatives thereof that provide protein-reactive functional groups.Of particular interest are para-alkoxy analogs of EDDP.

Also of interest are EDDP analogs covalently conjugated to anothersubstance or substituent, particularly through an alkoxy substituent inthe para position, and optionally through a linker group. Of particularinterest are proteins such as those used in immunogens or inenzyme-based immunoassays. However, this invention also embodies otherusefull EDDP conjugates, including but not limited to EDDP conjugated tocomplex carbohydrates; EDDP conjugated to labeling reagents such aschromophores, chemiluminescent and bioluminescent compounds, and stableand unstable isotypes; and EDDP conjugated directly or indirectly tosolid surfaces or particulates.

An EDDP analog or conjugate is said to be "purified" if it is (exceptfrom solvent) at least 50%, preferably at least about 90%, and even morepreferably at least about 99% pure when analysed by a suitable techniquesuch as GC/MS. A "synthetic" compound is a compound assembled fromcomponent parts by a process that does not involve live organisms orcells.

Various forms of EDDP and its analogs are interconvertible in aqueoussolution according to the following scheme: ##STR5## For the purposes ofthe present disclosure, these structures are considered to beequivalent. Where R is a substituent other than H, the compoundcomprises at least two chiral centers. The embodiments of this inventioninclude all stereoisomers, tautomers, salts, and protonated anddeprotonated forms of the structures shown unless otherwise indicated.

Certain preferred EDDP derivatives can be prepared by reactingp--HO--EDDP with a compound of the structure X-R⁰ or X=(R⁰)₂, where X isa leaving group. Preferred leaving groups are the halides Cl, Br, and I.This leads to ethers in which R is of the form R⁰ --O--. In someembodiments, R⁰ --is of the form Y--R¹ -- wherein R¹ is a hydrocarbyldiradical. Preferably, R¹ is a branched or unbranched hydrocarbyldiradical of 1-10 carbon atoms, or an alicyclic or aromatic hydrocarbyldiradical of 3-10 carbon atoms, or a combination thereof. Linear orbranched alkyl diradicals are preferred, especially of the structure--(CH₂)_(m) -- where m is 1 to 10, and particularly --(CH₂)--.

The substituent Y can have any structure of interest. Non-limitingexamples include those wherein Y is H, --COOH, an ester of the form--COOR², an amide of the form --CONHR^(A) or --CONR^(A) R^(B), athioether or thioester, any one of which may be further derivatized withother functional groups or conjugated to other substitutents,particularly protein, optionally through a linker group.

Where Y is --COOR², R² is typically a hydrocarbyl radical of 1-20 carbonatoms, preferably a branched or unbranched hydrocarbyl radical of 1-20carbon atoms, or an alicyclic or aromatic hydrocarbyl radical of 3-20carbon atoms, or a combination thereof. Linear or branched alkyidiradicals are preferred. t-butyl and other carboxylic acid protectinggroups are especially preferred.

In one example, treatment of p--HO--EDDP with p--t-butyl bromoacetateand KOH in DMSO gives the alkylated ether,p-(t-butyl-carboxymethoxy)--EDDP (p--t--Bu--CME--EDDP), shown in FIG. 7.Quantitative deprotection of p-(t-butyl-carboxymethoxy)--EDDP isachieved by stiring in trifluoroacetic acid for about 15 min followed bylyophilization (FIG. 8). The structure of the resulting p-carboxymethoxyEDDP (p--CME--EDDP) may be confirmed by ¹ H--NMR and mass spectroscopy.

A deprotected carboxyl group conjugated to the p-alkoxy position of EDDPin this fashion is one example of a protein reactive group that can beused to attach a protein of interest. The carboxylic acid derivativescan be condensed directly with amine groups on a protein, for example,in the presence of a carbodiimide to yield an amide bond conjugate.Alternatively, the carboxylic acid is first converted to an active esterand then condensed in a subsequent reaction with free amino groups onthe protein to yield an amide bond conjugate. In another procedure, anN-hydroxysuccinimide ester is first formed by reaction of the carboxylicacid with N,N-disuccinimidyl-carbonate (DSC). The active ester is thenreacted with the protein to yield an arnide bond conjugate. In yetanother procedure, the protein is conjugated to the p-carboxyalkoxy-EDDPusing 1-ethyl-3-(3-dimethyl aminopropyl)carbodiimide (EDC) andN-hydroxysuccinimide (NHS) in DMF.

In other preferred embodiments of this invention, the protein is linkedto an EDDP analog, not through an amide bond, but via a thioether bond.A maleimide adduct is first formed using an aminoalkyl-maleimidederivative. General synthesis of aminoalkyl-maleimide derivatives isdescribed by Huber et al. in PCT Application No. PCT/EP90/00957. Themaleimide adducts are then reacted with thiol groups on the protein togive a thioether-linked conjugate. Maleimide-sulfiydryl chemistry(Brinkley, Bioconjugate Chemistry 3:2-13, 1992) is generally more easyto control than amide bond condensation, allowing more exactingstochiometry of the conjugates which may be important for its intendedfunction.

Starting with a carboxylic acid analog of EDDP, condensation with amaleimidohydrocarbylamine (preferably maleimidoethylamine) afterpreactivation of the carboxylic acid with N-hydroxysuccinimide and acarbodiimide, preferably 1-ethyl-3-(3'-dimethylaminopropyl)-carbodiimide(EDC), yields a maleimide adduct in which Y in the formula recitedearlier has the general structure: ##STR6## R³ is preferably ahydrocarbyl diradical having 1-20 carbon atoms; either a branched orunbranched hydrocarbyl diradical of 1-20 carbon atoms, or an alicyclicor aromatic hydrocarbyl diradical of 3-20 carbon atoms, or a combinationthereof Linear or branched alkyl diradicals are preferred, especially ofthe structure --(CH₂)_(n) -- where n is 1 to 10, and particularly --CH₂CH₂ -- (in the case of maleimidoethylamine) or --(CH₂)₅ -- (in the caseof maleimidopentylamine).

One illustration is shown in FIG. 9. p--CME--EDDP is coupled withmaleimidoethylamine (MEA) usingO-benzotriazol-1-yl-N,N,N',N'-tetramethyluronium hexafluorophosphate(HBTU) and 1-hydroxybenzotriazole hydrate (HOBt) in acetonitrile toyield p-(maleimidoethylamino-carbonylmethoxy) EDDP (p--MEA--CME--EDDP),which can be confirmed by ¹ H--NMR.

A protein conjugate can then be prepared by combining an excess of themaleimide adduct with a protein having free thiol groups. Freesulfhydryls may be provided in the form of free cysteine residues or byreducing protein disulfide bonds by a reagent such as dithiothreitol.Alternatively, thiol groups can be added to a protein having freeprimary amino groups by reacting with 2-iminothiolane (IT) in aqueousbuffer, followed by removal of unreacted IT. A detailed protocol for thethiolation of the protein KLH is provided in U.S. Pat. No. 5,439,798.

The linkers of this invention are diradicals bonded in one position witha protein and in another position with EDDP analogs. Linkers aretypically organic diradicals comprising 1-30 carbons and optionally O,N, and S, and may be aliphatic, alicyclic, aromatic, or any combinationthereof. Certain preferred linkers are one or more alkyl diradicalsbonded to each other, and to the peptide, and to the p--HO--EDDP orp-carboxyalkoxy-EDDP through any combination of ether, ester and amidelinkages. Other preferred linkers are maleimidoalkylamine linkers of thegeneral form: ##STR7## wherein R⁴ has similar features to thosedescribed earlier for R³.

The composition of the substituents R¹, R³, and R⁴ are generally chosenaccording to the desired features of the EDDP conjugate that is theobject of the preparation. For example, where EDDP is coupled to a solidsupport or intact labeling agent, it may be desirable to use longersubstituents in order to improve accessibility of the EDDP core.Coupling to enzyme donor polypeptides in a cloned enzyme donorimmunoassay typically involve maleimidoethylamine adducts wherein R¹ ismethylene, so that antibody binding will inhibit association with theenzyme acceptor. When preparing an immunogen, the substituents R1, R3,and R4 are generally as short as possible so as not to alter solubilitycharacteristics and mininize the number of antibodies generated againstthe linker rather than EDDP. "Poly(amino acids)", "proteins","peptides", and "polypeptides" are terms used interchangeably herein todescribe polymers of amino acids of any sequence, typically at least 5amino acids in length, linked by peptide bonds. Of particular interestare proteins that can be used as immunogenic carriers, and proteins thatprovide a detectable signal for assay purposes, particularly enzymes andenzyme donor polypeptides.

For the purposes of obtaining specific antibodies against EDDP, anEDDP-protein conjugate of this invention will comprise a plurality ofEDDP analogs covalently bonded to an immunogenic protein carrierselected for its ability to provide a general immunostimulatory effect.Various protein carriers may be employed, including serum albumin, serumglobulins, ocular lens proteins, lipoproteins, ovalbumin, thyroxinebinding globulin, and synthetic polypeptides. Keyhole limpet hemocyanin(KLH) is especially preferred. KLH immunogens can be prepared by directcondensation with carboxyalkoxy-EDDP (e.g., KLH--p--CME--EDDP).Alternatively, KLH can be thiolated to provide thiol groups, forexample, with IT, and then coupled with maleimidoalkylamine (e.g.,KLH--IT--p--MEA--CME--EDDP).

The term "antibody" as used in this disclosure refers to both polyclonaland monoclonal antibody. The ambit of the term deliberately encompassesnot only intact immunoglobulin molecules, but also such fragments andderivatives of immunoglobulin molecules as may be prepared by techniquesknown in the art, and retaining the antibody activity of an intactimmunoglobulin. In this context, "antibody activity" refers to theability of an antibody to bind a specific antigen in preference to otherpotential antigens via the antigen combining site located within avariable region of an immunoglobulin. Fragments and other derivatives ofimmunoglobulins can be prepared by methods of standard proteinchemistry, such as subjecting the antibody to cleavage with aproteolytic enzyme like pepsin, papain, or trypsin; and reducingdisulfide bonds with such reagents as dithiothreitol. Geneticallyengineered variants of intact immunoglobulin can be produced byobtaining a polynucleotide encoding the antibody, and applying thegeneral methods of molecular biology to splice encoding sequences orintroduce mutations and translate the variant. Antibodies that areengineered variants of particular interest include chimeric andhumanized antibodies, Fab-like fragments, single-chain variable regionfragments (scFv), and diabodies.

For general techniques used in raising, purifying and modifyingantibodies, and the design and execution of immunoassays, the reader isreferred to Handbook of Experimental Immunology (D. M. Weir & C. C.Blackwell, eds.); Current Protocols in Immunology (J. E. Coligan et al.,eds., 1991); David Wild, ed., The Immunoassay Handbook (Stockton PressN.Y., 1994); and R. Masseyeff, W. H. Albert, and N. A. Staines, eds.,Methods of Immunological Analysis (Weinheim: VCH Verlags gesellschaftmbH, 1993).

Polyclonal antibodies of this invention are raised by administration ofthe immunogenic EDDP-protein conjugate to a mammalian host, usuallymixed with an adjuvant. The immunogen is conveniently prepared forinjection by rehydrating lyophilized immunogen to form a solution orsuspension. Preferred adjuvants are water-in-oil immersions,particularly Freund's complete adjuvant for the first administration,and Freund's incomplete adjuvant for booster doses. The preparation istypically administered in a variety of sites, and typically in two ormore doses over a course of at least 4 weeks. Serum is harvested andtested for the presence of EDDP antibody using an EDDP-protein conjugateor other EDDP analog in a standard immunoassay or precipitationreaction.

Polyclonal antisera will typically contain antibodies not reactive withEDDP and anti-EDDP antibodies cross-reactive with other substancesincluding methadone. Methods for purifying specific antibodies from apolyclonal antiserum are known in the art. A particularly effectivemethod is affinity purification using a column of EDDP conjugated to asolid phase. One manner of preparing an EDDP column is to conjugate EDDPto a protein other than the protein used in the immunogen, and thenattach the conjugate to a commercially available activated resin, suchas CNBr-activated SEPHAROSE™. The anti-EDDP is passed over the EDDPcolumn, the column is washed, and the antibody is eluted with a milddenaturing buffer such as 0.1 M glycine, 0.2 M NaCl, pH 2.5. If theanti-EDDP is passed over the column in a buffer containing freemethadone, then the bound and eluted fraction will be enriched forantibodies that are EDDP specific and don't cross-react with methadone.

Monoclonal antibodies of this invention can be prepared by a number ofdifferent techniques known in the art. For hybridoma technology, thereader is referred generally to Harrow & Lane (1988), U.S. Pat. Nos.4,491,632, 4,472,500, and 4,444,887, and Methods in Enzymology, 73B:3(1981). The most common way to produce monoclonal antibodies is toimmortalize and clone a splenocyte or other antibody-producing cellrecovered from an animal that has been immunized against EDDP asdescribed earlier. The clone is immortalized by a procedure such asfusion with a non-producing myeloma, by transfecting with Epstein BarrVirus, or transforming with oncogenic DNA. The treated cells are clonedand cultured, and clones are selected that produce antibody of thedesired specificity. Specificity testing is performed on culturesupernatants by a number of techniques, such as using the immunizingantigen as the detecting reagent in an immunoassay. A supply ofmonoclonal antibody from the selected clone can then be purified from alarge volume of culture supernatant, or from the ascites fluid ofsuitably prepared host animals injected with the clone. The antibody maybe tested for activity as raw supernatant or ascites, and is optionallypurified using standard biochemical preparation techniques such asammonium sulfate precipitation, ion exchange chromatography, and gelfiltration chromatography.

Alternative methods for obtaining monoclonal antibodies involvecontacting an immunocompetent cell or viral particle with anEDDP-protein complex of this invention in vitro. In this context,"immunocompetent" means that the cell or particle is capable ofexpressing an antibody specific for the antigen without further geneticrearrangement, and can be selected from a cell mixture by presentationof the antigen. Immunocompetent eukaryotic cells can be harvested froman immunized mammalian donor, or they can be harvested from anunimmunized donor and prestimulated in vitro by culturing in thepresence of immunogen and immunostimulatory growth factors. Cells of thedesired specificity can be selected by contacting with the immunogenunder culture conditions that result in proliferation of specific clonesbut not non-specific clones. Immunocompetent phage may be constructed toexpress immunoglobulin variable region segments on their surface. SeeMarks et al., New Engl. J. Med. 335:730, 1996; WO patent applications94/13804, 92/01047, 90/02809; and McGuinness et al., Nature Biotechnol.14:1149, 1996. Phage of the desired specificity may be selected, forexample, by adherence to an EDDP-protein complex attached to a solidphase, and then amplified in E. coli.

Antibodies obtained using any of the aforementioned techniques arescreened or purified not only for their ability to react with EDDP, butfor a low cross-reactivity with potential interfering substances. "Crossreactivity" is determined in a quantitative immunoassay by establishinga standard curve using known dilutions of the target analyte, EDDP. Thestandard curve is then used to calculate the apparent concentration ofthe interfering substance present in various known amounts in samplesassayed under similar condition. The cross reactivity is the apparentconcentration divided by the actual concentration multiplied by 100. Thepreferred immunoassay for determining cross-reactivity is a CEDIA® typeassay using an ED28-p--MEA--CME--EDDP donor polypeptide, described indetail in Example 4.

Acceptable levels of cross-reactivity of an anti-EDDP antibody formethadone are less than 5%, preferably less than 2%, more preferablyless than about 1%, more preferably less than about 0.5%, even morepreferably less than about 0.1%, and still more preferably less thanabout 0.02%. The antibody is also preferably less than 10%, morepreferably less than 2%, more preferably less than 0.5% cross-reactivewith EMDP. For purposes of monitoring methadone treatment,cross-reactivity with EMDP is generally less a concern than methadone.Since EMDP (like EDDP) is a naturally occurring methadone metabolite,its presence in a biological sample indicates compliance with treatmentrather than sample tampering. It is generally worth screening antibodiesfor cross reactivity with other pharmaceutical compounds that subjectsmay be taking collaterally, particularly those eliminated in urine andhaving a polycyclic structure with some resemblance to EDDP. Relevantcompounds include phenytoin and phenobarbital. Other compounds that weretested during the development of this invention are listed in Example 4.Levels of cross-reactivity for interfering compounds of this nature arepreferably less than 0.1%, more preferably less than 0.01%.

Selection of a particular antibody for a specific use involvesconsiderations more than just its cross-reactivity with potentialinterfering substances. Also relevant are such features as the reactionrates and affinity of the antibody which affect sensitivity and capacityof the reaction system, and the titer of antibody formed by a biologicalsource. Ultimate selection may require a compromise between thesevarious features.

Embodied in this invention are assay methods for the presence of EDDPand EDDP analogs in a sample of interest, including but not limited tosubjects suspected of being administered methadone and relatedcompounds, particularly humans. Suitable samples include biologicalsamples (particularly urine and serum) taken from subjects, optionallydiluted or modified to facilitate the assay, experimental samplesgenerated by any chemical or biological method, and standards containingknown concentrations of EDDP or other substances used for assaycalibration.

In most instances, the assays will involve using an antibody raisedagainst an EDDP-protein conjugate of this invention or having thecharacteristics of such an antibody, particularly a low cross-reactivitywith methadone itself.

The procedure entails combining the sample with the antibody underconditions that permit the formation of a stable complex between thesubstance to be tested (described herein as the "analyte", and typicallyEDDP), and the antibody. This is followed by detecting any EDDP-antibodycomplex that is formed. A "stable complex" is a complex between antibodyand analyte (typically non-covalently associated) that persists at leastas long as it takes the presence of the complex to be measured by theintended method.

The antibodies and EDDP conjugates of this invention may be implementedin any assay method known in the art.

Assays of this invention include both qualitative and quantitativeassays. Typical quantitative methods involve mixing the analyte with apredetermined non-limiting amount of the reagent antibody, andcorrelating the amount of complex formed with the amount of analyte inthe original sample using a relationship determined using standardsamples containing known amounts of analyte in the range expected forthe sample to be tested. In a qualitative assay, sufficient complexabove or below a threshold level established by samples known to containor be free of analyte establish the assay result. Unless otherwiserequired, "measuring" can refer alternately to qualitative andquantitative determination.

Assays of this invention include both separation-based and homogeneousassays. In separation based assays, the detecting of the complexinvolves a process wherein the complex formed is physically separatedfrom either unreacted analyte, unreacted antibody, or both. See, e.g.,U.S. Pat. No. 3,646,346. The complex may be first formed in the fluidphase, and then subsequently captured by a solid phase reagent orseparated on the basis of an altered physical or chemical property, suchas by gel filtration or precipitation. Alternatively, one of thereagents may be attached to a solid phase before contacting with otherreagents, and then the complex may be recovered by washing the solidphase free of unreacted reagents. Separation-based assays typicallyinvolve use of a labeled analog or antibody to facilitate detection orquantitation of the complex. Suitable labels are radioisotopes such as¹²⁵ I, enzymes such as peroxidase and β-galactosidase, and fluorescentlabels such as fluorescein isothiocyanate. The separation step involvesremoving labeled reagent present in complex form from unreacted labeledreagent. The amount of label in the complex can be measured directly orinferred from the amount left unreacted.

In homogeneous assays, the complex is typically not separated fromunreacted reaction components, but instead the presence of the complexis detected by a property which at least one of the reactants acquiresor loses as a result of being incorporated into the complex. Homogeneousassays known in the art include systems involving fluorochrome andfluorochrome quenching pairs on different reagents (U.S. Pat. Nos.3,996,345, 4,161,515, 4,256,834, and 4,261,968); enzyme and enzymeinhibitor pairs on different reagents (U.S. Pat. Nos. 4,208,479 and4,233,401); and chromophore and chromophore modifier pairs on differentreagents (U.S. Pat. No. 4,208,479). A preferred homogeneous assay systemis the cloned enzyme donor immunoassay, described in more detail below.

Assays of this invention include both sandwich and competition assays.Sandwich assays typically involve forming a complex in which the analyteto be measured is sandwiched between one reagent, such as a firstantibody used ultimately for separation of the complex, and anotherreagent, such as a second antibody used as a marker for the separatedcomplex. Competition assays involve a system in which the analyte to bemeasured competes with an analog of the analyte for binding to anotherreagent, such as an antibody. In the context of immunoassay, an "EDDPanalog" refers to a compound that is able to compete with EDDP forbinding to the antibody being used in the assay. EDDP analogs of thisinvention useful in immunoassay techniques include but are not limitedto EDDP-radioisotope conjugates, EDDP-enzyme conjugates, and otherEDDP-protein complexes. CEDIA® is an example of a competition assay. Theinvention also embodies assays that are neither sandwich nor competitionassays, as in certain assays involving immunoprecipitation.

Immunoassays specific for EDDP using anti-EDDP antibodies of thisinvention are rendered specific by virtue of the specificity of theantibody. For assays further employing EDDP-protein conjugates (such aswhen EDDP is labeled with an enzyme polypeptide), the EDDP can beattached to the protein conjugate by any suitable method. In certainpreferred embodiments, the chemistry described herein for formation ofpara-alkoxy analogs of EDDP is also used to prepare the EDDP-proteinconjugate used as an assay reagent. In this way, the EDDP core ispresented to the antibody in about the same orientation as during theimmunization event when the antibody was generated.

Assay methods of this invention are exemplified in the cloned enzymedonor immunoassay, described in U.S. Pat. No. 4,708,929. Relatedreagents and methods are taught in U.S. Pat. Nos. 5,254,577; 5,444,161;5,464,747; and 5,514,560. Cloned enzyme donor immunoassays forprocainamide and N-acetylprocainamide (NAPA) are described in U.S. Pat.Nos. 5,439,798 and 5,525,474. For the purposes of patent prosecution inthe U.S., the aforelisted patents are hereby incorporated herein intheir entirety. Cloned enzyme donor immunoassays are availablecommercially under the registered trademark CEDIA®. The reader isreferred to CEDIA® product inserts and technical manuals for furtherinformation.

Typically, a cloned enzyme donor immunoassay of this invention involvescombining the sample with: an EDDP-specific antibody; an enzyme donorpolypeptide conjugate; an enzyme acceptor polypeptide (wherein theenzyme acceptor polypeptide is capable of forming with said enzyme donorpolypeptide conjugate an active enzyme complex in the absence of anantibody to EDDP), and a substrate capable of being transformed by theactive enzyme complex into a product. The amount of product is thenmeasured, usually as a function of time.

The EDDP-specific antibody is preferably an antibody raised against anEDDP-protein conjugate of this invention, or having the characteristicsof such an antibody, especially a low level of cross-reactivity withmethadone. The antibody is also selected on the basis of three othercriteria. One, referred to as "inhibition", relates to how well theantibody binds the enzyme-donor conjugate. Sufficient inhibition(preferably at least about 70%) is needed in order to provide anadequate signal. A second criterion is the titer of the antibodyrequired to obtain the desired level of inhibition. Inhibition at lowerantibody levels is preferred. A third criterion, referred to as"modulation", relates to how well the sample analyte is able to competewith the conjugate for enzyme binding. Modulation is calculated as thedifference in enzyme rate between a sample having the analyte at atarget concentration (moderately chosen within the intended workingrange; preferably 100 ng/mL in the case of EDDP) and a sample having noanalyte, divided by the rate at the target concentration. Bettermodulation correlates with better assay sensitivity.

The enzyme-donor enzyme-acceptor pair is a pair of polypeptides whichspontaneously assemble in reagent buffer to form an active enzymecomplex. The active enzyme complex is capable of enzymaticallytransforming a substrate into a product that is differentiallydetectable. Typically, the product is a different color from thesubstrate and can be quantified in a spectrophotometer. The donor andacceptor pair are typically two functional subunits of a common enzyme.The subunits may be noncovalently associated in the native enzyme, orthey may be defective versions of a common polypeptide that complementeach other when together.

Preferred enzyme-donor and enzyme-acceptor polypeptides are based on theenzyme β-galactosidase polypeptide. A "β-galactosidase polypeptide" is apolypeptide identifiable on the basis of its amino acid sequence orenzymatic activity as being developed from an enzyme withβ-galactosidase activity. The definition encompasses not only naturallyoccurring β-galactosidase, but also fragments, deletion mutants, fusionproteins, mutants, and other variants based thereupon obtained by suchprocesses as enzymatic fragmentation and genetic engineering of relevantencoding sequences. Particular β-galactosidase polypeptides aredescribed in the aforelisted U.S. patent applications pertaining tocloned enzyme donor immunoassays.

β-galactosidase enzyme acceptors are preferably produced by a deletionmutant of the β-galactosidase gene. EA22, one of the preferredacceptors, has a deletion of amino acid residues 13-40. Other enzymeacceptor fragments of β-galactosidase which contain the natural sequencewhich includes amino acid position 602 may also be used. Other examplesinclude EA5, EA11, EA14, EA17, EA18, EA20, EA23 and EA24. The distal endof the deleted segment normally falls between amino acid positions 26and 54 of the β-galactosidase sequence. In EA22, the distal end of thedeletion segment is amino acid 40.

A particularly preferred β-galactosidase enzyme donor is ED28. This is afragment of β-galactosidase consisting of amino acids 1-46, withcysteine residues at positions 1 and 46. ED28 is described in EuropeanPatent Application No. 90308937.3. The two cysteine residues can be usedfor exact and reproducible placement of maleimide adducts of EDDP asdescribed earlier. Before conjugation with EDDP, reducing reagent thatis generally used in the storage of ED28 is removed by a suitabledesalting technique, such as on a Pharmacia NAP5™ column as described inU.S. Pat. No. 5,439,798. The EDDP is then conjugated with the maleimideadducts as described elsewhere in this disclosure. Adjustment of thelinkage can be performed by monitoring enzyme inhibition by anEDDP-specific antibody. Typical linker groups used are maleimide adductshaving the structure indicated earlier, wherein R⁴ is --(CH₂ CH₂)--.

Preferred substrates for use in immunoassays based on β-galactosidaseinclude those described in U.S. Pat. Nos. 5,032,503; 5,254,677;5,444,161 and 5,514;560. Amongst the preferred substrates ischlorophenol B--D-red galactopyranoside. Optimization of other featuresand conditions of the assays embodied by this invention is a matter ofroutine experimentation within the skill of the ordinary artisan.

Reagents and buffers used in the assays of this invention can bepackaged separately or in combination into kit form to facilitatedistribution. The reagents are provided in suitable containers, andtypically provided in a package along with written instructions relatingto assay procedures.

Further illustration of the development and use of antibodies and assaysaccording to this invention are provided in the Example section below.The examples are provided as a further guide to a practitioner ofordinary skill in the art, and are not meant to be limiting in any way.

EXAMPLES Example 1

Synthesis of racemic1,5-dimethyl-3-(p-methoxyphenyl)-3-phenyl-2-pyrrolidone

General Procedures

p-Methoxybenzophenone and 2-dimethyl-aminoisopropyl chloridehydrochloride were purchased from Aldrich Chemical Co. Melting points(m.p.) were determined on a Hoover capillary apparatus and areuncorrected. The IR spectrum was recorded on a Shimadsu IR-460spectrophotometer. ¹ H--NMR spectra were obtained on a Bruker WM-250 NMRspectrometer. Analytical precoated TLC plates (5×10 cm) were purchasedfrom Merck. The TLC plates were visualized using phosphomolybdic acidand then Ce(SO₄)₂ in sulfuric acid spray reagents.

p-methoxybenzhydryl alcohol

Solid NaBH₄ (2.55 g, 0.067 mol) was added portion-wise to a solution ofp-methoxybenzophenone (25.14 g, 0.118 mol) in MeOH (150 mL) at roomtemperature over 10 min. After the exothermic reaction had subsided, thereaction mixture was stirred at room temperature for 2 h. TLC (SiO₂,CHCl₃) indicated incomplete reaction; therefore, additional NaBH₄ (2.55g) was added and the reaction mixture was stirred at room temperature anadditional 2 h. The MeOH was evaporated and the residue partitionedbetween water (100 mL) and CH₂ Cl₂ (100 mL). The layers were separated,and the aqueous layer was extracted with CH₂ Cl₂ (200 mL). The combinedorganic layers were dried (Na₂ SO₄), filtered and evaporated to give thealcohol (25.24 g, 99%) as a white solid: m.p. 66-67° C. The structurewas confirmed by ¹ H--NMR in CDCl₃.

p-methoxybenzhydryl chloride

A stirred suspension of p-methoxybenzhydryl alcohol (25.04 g, 0.117 mol)and CaCl₂ (38.22 g, 0.344 mol) in benzene (400 mL) was bubbled withhydrogen chloride gas for 45 min. Afterwards, the mixture was stirred anadditional 1 h and then filtered. The filtrate was evaporated to givethe chloride (27.10 g, 99%) as an off-white solid. The structure wasconfirmed by ¹ H--NMR in CDCl₃.

2-(p-methoxyphenyl)-2-phenylacetonitrile

To a solution of p-methoxybenzhydryl chloride (27.00 g, 0.116 mol) indry CH₃ CN (200 mL) was added dibenzo-18-crown-6 (2.12 g) followed bydry KCN (8.02 g, 0.123 mol), and the stirred mixture was refluxed for 66h. After this time, the reaction mixture was cooled to room temperatureand the solid was removed by filtration. The filtrate was evaporatedunder vacuum. The resulting solid was dissolved in CH₂ Cl₂ (50 mL) andthe solution flushed through a silica gel column (230-400 mesh, 60 g)using CH₂ Cl₂. The eluent was evaporated and the residue was tituratedwith heptane to precipitate the product. The precipitate was collectedand washed with heptane to give a solid. ¹ H--NMR analysis of this solidshowed that it was a mixture of starting chloride and product.Therefore, the heptane filtrates were evaporated and the solids wererecombined, giving a total of 25.03 g of material. This solid wasreacted again as described above, except that the reflux period wasextended 48 h. Afterwards, the reaction mixture was worked up asdescribed above to give the title nitrile (18.56 g, 72%) as a yellowishwhite solid. The structure was confirmed by ¹ H--NMR in CDCl₃.

4-dimethylamino-2-p-methoxyphenyl)-2-phenylpentane-nitrile

The THF used in this procedure was freshly distilled frombenzophenone/Na metal, and the glassware was oven-dried at 120° C. for 4h. To a 1000 mL three neck round-bottom flask flushed with nitrogen wasadded THE (30 mL). The flask was cooled in an ice bath, then 2.5Mn-butyllithium in hexane (29.6 mL, 0.074 mol) was added by syringefollowed by diisopropylamine (7.48 g, 0.074 mol) to give a yellowsolution. To the cold stirred solution was added a solution of2-(p-methoxyphenyl)-2-phenylacetonitrile (15.00 g, 0.0672 mol) in THF(300 mL) drop-wise over 15 min to give a dark yellow solution. Then asolution of 2-dimethylaminoisopropyl chloride (35.95 g, 0.294 mol) inTHF (40 mL) was added and the reaction mixture was heated to gentlereflux under nitrogen. After refluxing for 18 h, the reaction wascomplete by TLC (SiO₂, CHCl₃). The reaction mixture was cooled to roomtemperature then evaporated under vacuum to an oily residue. The residuewas partitioned between ether (300 mL) and water (300 mL). The layerswere separated and the ether layer was washed with saturated NaCl (300mL), then dried (Na₂ SO₄). The ether was filtered, and the filtrateevaporated under vacuum to give a viscous yellow oil (22.55 g). The oilwas chromatographed on silica gel (230-400 mesh, 1000 g) using 1%acetone/CHCl₃ (6000 mL), 2% acetone/CHCl₃ (4000 mL), 3% acetone/CHCl₃(1000 mL), 5% acetone/CHCl₃ (8000 mL), 10% acetone/CHCl₃ (7000 mL) andacetone (3000 mL) to give the title nitrile (13.53 g, 65%) as a viscousyellow oil. The structure was confirmed by ¹ H--NMR in CDCl₃.

4-(N-2,2,2-trichlorocarboethoxy-N-methylamino)-2-(p-methoxyphenyl)-2-phenylpentanenitrile

A stirred solution of4-dimethylamino-2-(p-methoxyphenyl)-2-phenylpentanenitrile (13.43 g,0.044 mol) in toluene (100 mL) was heated to reflux under nitrogen and asolution of 2,2,2-trichloroethylchlorofornate (10.70 g, 0.051 mol) intoluene (50 mL) was added drop-wise over 30 min. The reaction mixturewas refluxed for 24 h. TLC [SiO₂, CHCl₃ :MeOH:conc. NH₄ OH (90/10/4drops per 100 mL)] indicated incomplete reaction; therefore, additional2,2,2-trichloroethylchloroformate (5.35 g) was added and the reactionmixture was refluxed another 24 h. After this time, the reaction mixturewas cooled to room temperature and treated with 88% formic acid (6 mL)followed by triethylamine (14 mL), added slowly. Afterwards, water (200mL) was added and the layers were separated. The aqueous layer wasextracted with ether (200 mL). The organic layers were combined andwashed with 10% HCl (2×250 mL) and saturated NaCl (200 mL). The organiclayer was dried (Na₂ SO₄) and evaporated under vacuum to obtain aviscous yellow oil (23.04 g). The oil was chromatographed on silica gel(230-400 mesh, 500 g) using CHCl₃ to give a title nitrile (18.60 g, 91%)as a viscous yellow oil. The structure was confirmed by ¹ H--NMR inCDCl₃.

2-imino-1, 5-dimethyl-3-(p-methoxyphenyl-3-phenyl-pyrrolidinehydrochloride

A solution of4-(N-2,2,2-trichloro-carboethoxy-N-methylamino)-2-(p-methoxyphenyl)-2-phenyl-pentanenitrile(18.48 g, 0.039 mol) in dry DMF (150 mL) was cooled in an ice bath undernitrogen for 30 min. To the cold stirred solution was added 95% formicacid (4.3 g) followed by Zn dust (5.5 g) and the reaction mixture wasstirred at room temperature for 48 h. Afterwards it was filtered,washing the solid with CH₂ Cl₂ (100 mL). The filtrate was evaporatedunder vacuum using a vacuum pump to give an oil. The oil was dissolvedin CHCl₃ (200 mL) and the solution was washed with 10% NH₄ OH (2×100mL). The combined aqueous layers were extracted with CHCl₃ (100 mL). TheCHCl₃ layers were combined, dried, (Na₂ SO₄) and evaporated under vacuumto obtain a viscous yellow oil (12.47 g). The oil was dissolved in ether(400 mL) and the solution was extracted with 10% HCl (2×100 mL). Theaqueous layers were combined and extracted with ether (2×200 mL) andthen with CHCl₃ (3×100 mL). The CHCl₃ extracts were combined, dried (Na₂SO₄) and evaporated to give the crude imine hydrochloride (11.36 g, 87%)as a tan foam. The structure was confirmed by ¹ H--NMR in CDCl₃.

1,5-dimethyl-3-(p-methoxyphenyl)-3-phenyl-2-pyrrolidone

A solution of crude2-imino-1,5-dimethyl-3-(p-methoxy-phenyl)-3-phenylpyrrolidinehydrochloride (11.26 g, 0.034 mol) in 10% HCl (500 mL) was heated to asteam bath. While heating on the steam bath, KNO₂ (100 g) was addedportion-wise over 30 min. CAUTION! The reaction foams with eachaddition. Following the addition of KNO₂ the reaction mixture was heatedon the steam bath for 30 min. Additional 10% HCl (100 mL) was addedfollowed by another portion-wise addition of KNO₂ (50 g), after whichthe reaction mixture was heated for 30 min. Afterwards, the reactionmixture was cooled to room temperature and extracted with ether (3×200mL). The combined ether layers were dried (MgSO₄) and evaporated undervacuum to obtain a viscous yellow oil (7.78 g). The oil waschromatographed on silica gel (230-400 mesh, 400 g) using CHCl₃ to givethe title compound (5.99 g, 70%) as a viscous yellow oil. The structurewas confirmed by ¹ H--NMR in CDCl₃, and characterized by IR in CHCl₃.The final yield after additional drying, characterization and transferwas 4.87 g.

Analysis

Calculated for C₁₉ H₂₁ NO₂ •CHCl₃ : C, 75.93; H, 7.04; N, 4.65. Found:C, 76.15; H, 7.19; N, 4.72.

Example 2

Synthesis of racemic2-ethyl-1.5-dimethyl-3-p-hydroxyphenyl-3-phenylpyrroline hydrochloride

(+/-)-1, 5-dimethyl-3-(-hydroxyphenyl)-3-phenyl-2-pyrrolidone

(+/-)-1,5-dimethyl-3-(p-hydroxyphenyl)-3-phenyl-2-pyrrolidone (fromExample 1), 1.5 g, was dissolved in 30 mL dichloromethane. The flask wasflushed with argon and chilled in an acetone/dry ice bath to -60° C. I Mboron tribromide in dichloromethane, 10 mL, was then added drop-wiseover 18 min with stirring. The reaction was allowed to come to roomtemperature and stirred for 22 h. Methanol, 10 mL, was then added slowlyto quench the reaction. The resultant solution was rotary evaporated togive an oil redissolved in 10 mL methanol and rotary evaporated a secondtime. The oil was diluted with 20 mL water to give a precipitate. 6 Nhydrochloric acid, 6 mL, was added followed by 50 mL dichloromethane.Mixing gave a biphasic solution. The phases were separated and the upperaqueous phase was reextracted twice with 25 mL portions ofdichloromethane. The combined dichloromethane extracts were washed withsaturated sodium chloride solution, dried over sodium sulfate, andevaporated to give a foam residue.

The product was crystallized from diethyl ether to give 1.0 g. m.p.164-170° C. (uncorrected); TLC: Chlorofoinlmethanol (9:1) silica 60F254--single UV absorbing and iodine staining spot at Rf0.15. ¹H--NMR(CDCl₃): 1.26 ppm (d,d), 5 CH₃ ; 2.2 and 2.9 ppm (ms), 4 CHs; 2.89ppm(s), 1 N--CH₃ ; 3.5 ppm (m), 5 CH; 6.1 ppm (d), p--HO; 6.6 and 7.1ppm (dds), p--HO-phenyl; 7.3 ppm (m), phenyl.

(+/-)-1,5-dimethyl-3-(p-tert-butoxyphenyl)-3-phenyl-2-pyrrolidone

(+/-)- 1 ,5-dimethyl-3-(p-tert-butoxyphenyl)-3-phenyl-2-pyrrolidone, 516mg, was suspended in 12 mL of dichloromethane. The flask was purged withargon. Concentrated sulfuiric acid, 20 μl, was then added andisobutylene was bubbled in slowly through a sparging tube. After one han additional 9 mL of dichloromethane and 10 μl of sulfturic acid wereadded, and the flask was stoppered and allowed to stand at roomtemperature for 18 h. The reaction mixture was then diluted with 10 mLwater and mixed to obtain a biphasic solution. The phases were separatedand the lower dichloromethane phase was washed with saturated sodiumchloride solution, dried over sodium sulfate and rotary evaporated togive an oil. The crude product contained some starting material and sideproduct.

Purification by silica gel flash chromatography in acetone/chloroform(9:1) gave 454 mg of an oily product. TLC: chloroform/acetone 9:1;silica 60 F254, single UV absorbing and iodine staining spot Rf0.5;1H--NMR (CDCl₃ : 1.27 ppm (m) 5-CH3 overlapped with 1.32 ppm (s) t--BuCH3s; 2.2 and 3.0 ppm (ms), 4 CHs: 2.9 ppm (s) 1 N-CH3; 3.5 ppm (m), 5CH; 6.9 ppm (d) p--tBuO-phenyl; 7.2-7.4 ppm (ms), p--tBuO-phenyl andphenyl.

(+/-)-2-ethylidene-1,5-dimethyl-3-(p-tert-butoxyphenyl)-3-phenylpyrrolidine

Ethyl lithium was prepared fresh by weighing out 84 mg of lithium ribbonunder argon and transferring to a Schlenk flask equipped with a droppingfinnel plus magnetic stir bar and purged with argon. Anhydrous diethylether, 10 mL, was added to the flask. The flask was then cooled to -40°C. in an acetone/dry ice bath. In the meantime, a solution of 710 mg ofbromoethane in 8 mL ether was prepared and added to the dropping funnel.The bromoethane solution was added to the vigorously stirred lithiumsuspension over 5 min at -40° C. to -5° C. to give a turbid, nearlycolorless solution of ethyl lithium.

(+/-)-1,5-dimethyl-3-(p-tert-butoxyphenyl)-3-phenyl-2-pyrrolidone, 717mg, was then dissolved in 8 mL toluene and transferred to the droppingfunnel. The solution was added drop-wise to the stirred ethyl lithium at-5° C. over 5 min. The reaction was then allowed to warm up to room tempover 1 h. After 80 min at room temp, the reaction was quenched bydrop-wise addition of 10 mL ice water. The biphasic mixture wasseparated and the upper organic phase was washed with water followed bysaline solution. The organic phase was dried over magnesium sulfate,filtered and rotary evaporated to give 656 mg of crude product as amixture of cis and trans isomers.

TLC

n-butanol/acetic acid/ water 4:1:1; silica 60 F254; major UV absorbing,iodine staining spot at Rf0.42; minor spot at Rf0.50. ¹ H--NMR (CDCl₃):0.92 ppm (m), cis ethylidene CH₃ ; 1.09-1.15 ppm (ds) cis/trans 5-CH₃ ;1.31 ppm (s) & 1.37 ppm (s), cis/trans t--BuO CH₃ s; 1.7 ppm (m), transethylidene CH₃ ; 2.35 ppm (s) & 2.63 ppm (s), cis/trans 1-N--CH₃ s; 3.6ppm (m), trans ethylidene CH; 4.3 ppm (m), cis ethylidene CH; 6.8-7.5ppm (ms), ArH.

(+/-)-2-ethyl-1,5-dimethyl-3-(p-hydroxyphenyl-3-phenylpyrrolinehydrochloride (p--HO--EDDP.HCl)

(+/-)-2-ethylidene-2.5-dimethyl-3-(p-tert-butoxyphenyl)-3-phenylpyrrolidine,648 mg, was dissolved in 4 mL dioxane, purged with argon, then dilutedwith 4 mL of 4 N hydrogen chloride/dioxane reagent. The reaction wasstirred at room temp. for 3 h. The resultant orange red solution withsome reddish solids was filtered through a sintered glass funnel and thefiltrate was diluted with 50 mL diethyl ether to obtain an oily redprecipitate. The other was decanted and the oil was washed with freshether by sonication. The ether was decanted and the oil was dissolved in100 mL chloroform.

The solution was rotary evaporated in a taxed flask to give 311 mg ofproduct as a pale red-orange foam. ¹ H--NMR(CD₃ CN): Endocyclic doublebond; two diastereomers; 0.6 ppm (overlapping ts="d of d"), 2-ethyl CH₃1.55 ppm (d of d), 5-CH₃ ; 2.3-2.7 ppm (d of q), 4 CH; 2.75 ppm (q),2-ethyl CH₂ ; 3.0-3.3 (d of q), 4 CH; 3.5 ppm (2 s="d"), 1-N-CH₃ s; 4.5ppm (2 overlapping qs), 5 CH; 7.0-7.6 ppm, Ar--H Mass spec[M+H]=294.3=theory; HPLC : C4RP; 5 min at 16% CH₃ CN/20 mM triethylamineacetate pH 6 (TEA--Ac) followed by 16-46% CH₃ CH/20 mM TEA--Ac(0.75%/min); flow 1 mL/min; monitored at 234 & 252 nm: two peaks withk'=5.58 (major) and 6.19 (minor).

Example 3

Preparation of p--HO--EDDP adducts, immunogens, and conjugates

Alkylation of p--HO--EDDP

Freshly ground KOH (13.2 mg 200 μmoles) was stirred under 200 μl DMSOfor 5 min. p--HO--EDDP.HCl (13.2 mg. 40 μmoles), prepared as in Example2, was added, followed by t-butyl bromoacetate (25.4 mg. 120 μmoles).After stirring for 110 min 3 mL of 20 mM, trifluoroacetic acid (ITA) inwater was added followed by 4 μl additional neat TFA. The mixture waspurified in two injections by HPLC (1×25 cm C4 column; Buffer A=20 mMTFA in water, Buffer B=20 mM TFA in CH₃ NC; 0 min, 100% A; 0.1-10 min,25% to 35% B; 10.1-20 min, 45% to 50% B; flow rate=4 mL/min; 280 nm) ona 10 mL loop. Lyophilization yielded p--t-butyl-carboxymethoxy-EDDP(p--t--Bu--CME--EDDP) (14.5 mg 69%) as pale red droplets.

Deprotection of p--t--Bu--CME--EDDP

TFA (1 mL) was added to p--t--Bu--CME--EDDP (19.4 mg 37.1 μmole) withstirring. After stirring for 15 min at room temperature (RT) thesolution was frozen in liquid nitrogen and lyophilized to yieldp--CME--EDDP (18.5 mg, 107%, retains some TFA) as pale red droplets. ¹H--NMR (200 MHz, CD₃ CN) ppm 6.95-7.45 (9 H, m, ArH), 4.70 (2 H, s, OCH₂CON), 3.50 (3 H, s, CH₃ N) 3.1 & 3.3 (1 H @, 4-CH₂, dd, J_(a) =X Hz,J_(b) =X Hz), 0.59 (3 H, dt, J=2.4, 7.6 Hz, CH₃ CH₂) MS (M+,352.2).

Coupling of p--CME--EDDP with Maleimidoethylamine (MEA)

MEA--HCl (5.30 mg, 20 μmoles),O-benzotriazol-1-yl-N,N,N',N'-tetramethyluronium hexafluorosphosphate(HBTU) (7.59 mg, 15 μmoles), 1-hydroxybenzotriazole hydrate (HOBt) (2.70mg, 15 μmoles) and disopropylethylamine (DIEA) (12.9 mg, 100 μmoles)were added with stirring to p--CME--EDDP (4.66 mg, 10 μmoles) in 1 mLCH₃ CN. After an hour, 8 mL of 0.1% aqueous TFA were added to themixture. The resulting solution was purified in a single injection on a10 mL loop by HPLC (1×25 cm C4 column; 0 min, 100% A; 0.1-20 min, 10 to50% B; flow rate=4 mL/min; 260 nm). Lyophilization gavemaleimidoethylamino-carbonylmethyl ether-EDDP (p--MEA--CME--EDDP) (4 mg68%) as a hygroscopic red foam ¹ H NMR (200 MHz, CD₃ CN) ppm (minorisomer): 0.59 (3 H, t, J=7.6 Hz, 2-CCH₂ CH₃). 1.43 (3 H, d, J=6.8 Hz,5-CH₂ CH₃), 2.46 (1 H, dd, J=6.6, 7.4 Hz, 4-CH_(a) CH_(b)), 2.72 (2 H,m, 2-CCH₂), 3.15 (1 H, dd, J=6.6 Hz, 7.3 Hz, 4-CH_(a) CH_(b)), 3.41 (2H, t, J=5.9 Hz, CH₂ NCO 3.51 (3 H, s, CH₃ N); 3.59 (2 H, t, J=5.6 Hz,CH₂ NCO); 4.42 (2 H, s, OCH₂ CON); 6.72 (2 H, s, maleimide); 6.97-7.44(9 H, m, ArH); ppm (major isomer): 0.60 (3 H, t, J=7.6 Hz, 2-CCH₂ CH₃).1.47 (3 H, d, J=6.6 Hz, 5-CH₂ CH₃), 2.56 (1 H, dd, J=6.2, 7.7 Hz,4-CH_(a) CH_(b)), 2.72 (2 H, m, 2-CCH₂), 3.24 (1 H, dd, J=6.4 Hz, 7.7Hz, 4-CH_(a) CH_(b)), 3.41 (2 H, t, J=5.9 Hz, CH₂ NCO), 3.51 (3 H, s,CH₃ N), 3.59 (2 H, t, J=5.6 Hz, CH₂ NCO); 4.42 (2 H, s, OCH₂ CON); 6.76(2 H, s, maleimide); 6.97-7.44 (9 H, m, ArH).

Preparation of KLH--p--CME--EDDP Immunogen

1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) (8.63 mg. 45 μmoles)and N-hydroxysuccinimide (NHS) (5.18 mg, 45 μmoles) were added withstirring to p--CME--EDDP (3.50 mg 7.5 μmoles) in 1 mL DMF. After 2hours, 1 niL of phosphate buffer (100 mM, pH=7) was added, followed bykeyhole limpet hemocyanin (KLH) (15 mg) in 1.5 mL of phosphate buffer(83 mM, pH=7.2, 0.9 M NaCl). After stirring for 5 hours, the mixture wasdialyzed against 800 mL phosphate buffer (10 mM, pH=7, 150 mM NaCl) and200 mL DMF. After 12 hours, the buffer was replaced. After an additional12 hours, this buffer was replaced with 2 L of phosphate buffer (10 mM,pH=7, 150 mM NaCl) which was again replaced after another 12 hours.Twelve hours after the last buffer replacement, the immunogen wastransferred to a vial and stored at -80° C. until used.

Preparation of KLH-2-IT--p--MEA--CME--EDDP Immunogen

2-Iminothiolane (2-IT) (2.06 mg, 15 μmoles) and 3.5 mL of phosphatebuffer (100 mM, pH=8) were added to KLH (15 mg) in 1.5 mL of phosphatebuffer (83 mM, pH=7.2, 0.9 M NaCl) with stirring. After 75 min, themixture was split into two equal portions and each portion was desaltedwith a PD-10 pre-packed SEPHADEX™ G-25 ion exchange column (Pharmacia,Inc.) pre-equilibrated with phosphate buffer (100 mM, pH=8) to removeexcess 2-IT. The eluant was added to p--MEA--CME--EDDP (1.77 mg, 3μmoles) in 1.5 mL DMF. After stirring for 5 hours, the mixture wasdialyzed against 800 mL phosphate buffer (10 mM, pH=7, 150 mM NaCl) and200 mL DMF. After 12 hours, the buffer was replaced. After an additional12 hours this buffer was replaced with 2 L of phosphate buffer (10 mM,pH=7, 150 mM NaCl), which was again replaced after another 12 hours.Twelve hours after the last buffer replacement, the immunogen wastransferred to a vial and stored at -80° C. until used.

Conjugation of p--MEA--CME--EDDP to the enzyme donor ED28

A solution of desalted ED28 (1 mg, 102 nmol) in 234 μl phosphate buffer(100 mM, pH=7) was added with stirring to a solution ofp--MEA--CME--EDDP (361 μg 610 nmol) in 180 μl DMF. After standing at RTfor 1 hour, 586 ,μl of 20 mM TFA in water was added and the mixture wasdesalted on a PD-10 column pre-equilibrated with 20 mM TFA in water. Theeluant (1.5 mL) was injected in a 10 mL loop and purified by HPLC (C41×25 cm, 0 min, 100% A; 0.1-20 min, 25-45% B; flow rate=4 mL/min; 280nm). The total volume of eluant was 4.55 mL. The yield was 684 μg (61%)as determined by UV absorbance at 280 nm (ε₂₈₀ =22,080). This solutionwas stored at -80° C. until further use.

Example 4

Development of EDDP-specific monoclonal antibodies and immunoassays

Immunization and hybridoma production

Monoclonal antibodies specific for EDDP were obtained using theimmunogen KLH--p--CME--EDDP or alternativelyKLH-2-IT--p--MEA--CME--EDDP. A priming injection and two boosterinjections were given using the same immunogen for each mouse. A totalof 16 mice were immunized. The mice were bled, and each serum wereassayed in a 96-well plate immunoassay. The assay used was a clonedenzyme donor immunoassay method, using ED28-p--MEA--CME--EDDP, preparedas in Example 3. Results are shown in Table 1:

                                      TABLE 1                                     __________________________________________________________________________    Immunoassay of Mouse Sera                                                                        Modulation                                                                           Relative methadone cross-                             Average with free reactivity                                                Immunogen      Titer                                                                             EDDP   range average                                       __________________________________________________________________________    KLH-p-CME-EDDP 1:2500                                                                            62% ± 5%                                                                          0-9%   2%                                             (No Linker)                                                                   KLH-2-IT-p-MEA-CME-EDDP 1:1300 59% ± 2% 0-31% 17%                          (MEA linker)                                                                __________________________________________________________________________

Titration results showed very good titer, 89-90% inhibition, and about60% modulation with 10 X cut-off concentration. These results indicatethat both immunogens work well to elicit a strong and specific response.

A mouse from the zero-linker group was chosen as the source ofimmunocompetent cells for the first fusion because of a slightly highertiter and lower average cross-reactivity with methadone. The parentalmyeloma used for all fusions was P3X63-Ag8.653, purchased through theAmerican Type Culture Collection (ATCC). At the time of fusion, thespleen of the donor mouse was moderately enlarged. The first fusionproduced 179 clones that strongly bound the EDDP conjugate (>60%inhibition) in a 96-well assay. Forty-four of these were retained on thebasis of good modulation with EDDP and low cross-reactivity tomethadone. A second fusion was performed in a similar fashion, fromwhich 72 initial EDDP-binding positive clones were identified, of which18 were retained. Culture supernatants from retained lines were grown toprovide antibody samples for instrumentation analysis.

Assay development and antibody selection

Prototype imnunoassays for EDDP were developed using the cloned enzymedonor immunoassay technology described in U.S. Pat. No. 4,708,929. Theseassays were used to identify particular monoclonal antibodies withacceptable combinations of inhibition, titer, modulation, and lowcross-reactivity.

Assays were performed as follows: Three μL of calibrator, control, orurine sample were automatically pipetted into the cuvette of aBoehringer Mannheim/Hitachi 717 Automated Analyzer, followed by 130 μLof Reagent 1 containing the β-galactosidase enzyme acceptor EA22 (1.7g/L), a monoclonal antibody to EDDP (varying concentration), 0.5%(vol/vol) fetal bovine serum, and 10 mM magnesium acetate in assaybuffer at a final pH of 6.9. The assay buffer was 0.4 M NaCl, 0.1 M1,4-piperazinediethanesulfonic acid, 10 mM ethyleneglycol-bis-(β-aminoethyl ether) N,N,N',N'-tetraacetic acid and 26 mMNaN₃. This mixture was incubated at 37° C. for 5 minutes, followed byaddition of 130 μL Reagent 2. Reagent 2 contained the enzyme donorconjugate ED28-p--MEA--CME--EDDP, 2 mg/mL of the substratechlorophenol-red-β-D-galactopyranoside, 2 mg/mL pepsin-fragmented bovineserum albumin (U.S. Pat. No. 5,212,081) and 5 mMethylenediaminetetraacetic acid (EDTA) in assay buffer. Incubationcontinued at 37° C., and absorbance measurements were taken at 12-secondintervals beginning 4 minutes after adding Reagent 2. The analyzerautomatically measures the rate of change of absorbance at 570 nm,corrected for background absorbance at 660 nm.

The concentration of each antibody in the Reagent 1 formulation wasvaried to yield a standard curve exhibiting optimum response near theconcentration of 100 ng EDDP/mL. The concentration of EDDP in a controlor unknown sample can be estimated by comparing the rate of change ofabsorbance with the control or unknown sample with the rates of thecalibrators. In normal use, the rate obtained for an unknown urinesample would be compared to the rate obtained for 100 ng EDDP/mLcalibrator, termed the "cut-off" calibrator. Sample exhibiting ratesabove that of the cut-off calibrator are considered positive for EDDP,while those with rates below that of the cut-off calibrator areconsidered negative.

Antibody clones retained from the initial screening were tested for acombination of inhibition, titer, modulation, and low cross-reactivity,and a number were selected for further analysis.

A key goal of the assay was to achieve maximum discrimination betweenEDDP and methadone. Cross-reactivity to methadone was determined bytesting samples containing various concentrations of methadone, anddetermining the concentration of methadone in urine which would give arate equal to that of the cut-off EDDP calibrator (100 ng EDDP/mL).Cross-reactivity is determined by dividing this concentration ofmethadone by 100 ng/mL, and multiplying by 100%. Methadonecross-reactivities measured for several monoclonal antibodies to EDDPare as follows:

                  TABLE 2                                                         ______________________________________                                        Cross-reactivity of Monoclonal antibodies to EDDP                                         Relative cross reactivity to                                                                   Relative cross-reactivity to                       Antibody Methadone EMDP                                                     ______________________________________                                        6A9     0.47%            --                                                     14C4 0.37% --                                                                 15E11 0.61% --                                                                5B12 0.26% 1.5%                                                               14G4 0.03%  0.01%                                                             21G7 0.34% 1.5%                                                               2C11 -- 1.5%                                                                  9H3 -- 0.1%                                                                   13A6 -- 1.5%                                                                ______________________________________                                    

Five monoclonal antibodies were tested with a panel of about 50compounds that were either licensed pharmaceuticals or potentialsubstances of abuse, at levels that were high relative to theconcentration likely to occur in test samples. Compounds showingreaction rates above the EDDP cut-off sample were retested in a dilutionseries. Results are shown in the next Table. Levels not marked werenegative.

                  TABLE 3                                                         ______________________________________                                        Cross-reactivity of Monoclonal antibodies to EDDP                                               Monoclonal antibody                                         Compound          5B12   6A9    14C4 14G4 21G7                                ______________________________________                                        Pentazocine 2.17 mg/mL            +    +                                         1:10   +                                                                      1:100                                                                         1:1000                                                                       Phencyclidine 2.33 mg/mL + +  + +                                              1:10 +    +                                                                   1:100                                                                         1:1000                                                                       Loperamine 1.46 mg/mL   +                                                      1:10                                                                          1:100                                                                         1:1000                                                                       2-OH Imipramine 127 μg/mL                                                   1:10                                                                          1:100                                                                         1:1000                                                                       Dextromethorphan 1.83 mg/mL  +  +                                              1:10                                                                          1:100                                                                         1:1000                                                                       alpha-Methadol 1.57 mg/mL + + +  +                                             1:10 +                                                                        1:100                                                                         1:1000                                                                       d-Methamphetamine 3.33 mg/mL +    +                                            1:10                                                                          1:100                                                                         1:1000                                                                       Norpropoxyphene 560 μg/mL                                                   1:10                                                                          1:100                                                                         1:1000                                                                       Propoxyphene 2.53 mg/mL  +  +                                                  1:10                                                                          1:100                                                                         1:1000                                                                     ______________________________________                                    

FIG. 10 shows the cloned enzyme donor immunoassay standard curves formonoclonal antibody 14C4 (Panel A) and 14G4 (Panel B). The 14G4 standardcurve shows an inhibition of 65.1% over the open reaction rate of 285.6mAU/min, a saturation at 100 ng/mL of 21.4%, and a modulation at 100ng/mL of 28.5%.

Testing of biological samples

Prototype EDDP assays using various monoclonal antibodies to EDDP wereevaluated for specificity using urine samples known to be free ofmethadone and other drugs, and for sensitivity using samples fromsubjects on methadone maintenance and containing known concentrations ofmethadone. Methadone levels were determined independently using theCEDIA® DAU methadone assay kit which is commercially available. Resultsfor several monoclonal antibodies are summarized in the next Table.

                  TABLE 4                                                         ______________________________________                                        Assay results using biological test samples                                           Samples established by                                                                         Samples established by                                 immunoassay to be immunoassay to be                                           METHADONE-NEGATIVE METHADONE-POSITIVE                                               EDDP      EDDP       EDDP    EDDP                                       Antibody negative positive negative positive                                ______________________________________                                        5B12    46        0          1       109                                        6A9 45 1 0 66                                                                 14C4 45 1 0 66                                                                14G4 46 0 1 109                                                             ______________________________________                                    

Samples containing 300 ng/mL methadone by immunoassay were identified aspositive for methadone; samples containing less than this amount wereconsidered negative.

Twenty-four samples which tested positive by the EDDP immunoassay methodwere further evaluated for EDDP by gas chromatography/mass spectrometry(GC/MS). All 24 samples were confirmed to contain EDDP by GC/MS. The onesample testing positive for EDDP in the methadone-negative group wasconfirmed by GC/MS to be negative for methadone but having >100 ng/mLEDDP. In addition, 50 urine samples known to be opiate positive weretested in the EDDP assay using antibody 14G4. Only one tested positiveat the cut-off level of >100 ng/mL EDDP, and was vindicated ascontaining methadone with trace amounts of EDDP.

Monoclonal antibody 14G4 had the following characteristics: Of 110methadone-positive samples, 109 tested positive for EDDP, giving asensitivity of 99.1%. Specificity testing using 382 EDDP negativesamples was 100%. Average cross-reactivity for methadone at 1000 μg/mL:0.028%; at 100 μg/mL: 0.026%. The following drug-positive samples werealso tested using antibody 14G4: propoxyphene (n=46), cocaine (n=50),opiates (n=49), amphetamines (n=91), barbiturates (n=48), PCP (n=50),benzodiazepines (n=48). None tested positive for EDDP.

What is claimed as the invention is:
 1. A compound of the formula:##STR8## and salts thereof, wherein R comprises a poly(amino acid)attached through p--CO--L-- where L is a bond or a diradical linkergroup.
 2. A compound of the formula: ##STR9## and salts thereof, whereinR¹ is a hydrocarbyl diradical having 1-10 carbon atoms andY is--CO--L--Q, wherein Q is a poly(amino acid) and L is a bond or adiradical linker group.
 3. The compound of claim 2, wherein R¹ is alinear or branched alkyl diradical.
 4. The compound of claim 2, whereinL is ##STR10## wherein R⁴ is a hydrocarbyl diradical of 1-20 carbonatoms.
 5. The compound of claim 2, wherein L is a bond and Y is --CO--Q.6. The compound of claim 2, wherein R¹ is --(CH₂)_(m) -- and m is 1 to10.
 7. The compound of claim 2, wherein R¹ is --CH₂ --.
 8. The compoundof claim 4, wherein R⁴ is --(CH₂)_(n) -- and n is 1 to
 10. 9. Thecompound of claim 4, wherein R⁴ is --CH₂ CH₂ --.
 10. The compound ofclaim 2, wherein Q is an immunogenic poly(amino acid).
 11. The compoundof claim 2, wherein Q is keyhole limpet hemocyanin (KLH) or thiolatedKLH.
 12. The compound of claim 2, wherein Q comprises an enzyme or aportion thereof.
 13. The compound of claim 2, wherein Q is an enzymedonor polypeptide.
 14. The compound of claim 13, wherein the enzymedonor polypeptide is a β-galactosidase polypeptide.
 15. An antibodyraised against a compound according to claim 2 and specific for thecompound 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidone (EDDP). 16.An antibody raised against a compound according to claim 4 and specificfor EDDP.
 17. An antibody specific for EDDP and having across-reactivity with methadone of less than 2.0%.
 18. The antibody ofclaim 15, having a cross-reactivity with methadone of less than 0.5%.19. The antibody of claim 15, having a cross-reactivity with methadoneof less than 0.1%.
 20. An antibody specific for EDDP and having across-reactivity with the compound2-ethyl-5-methyl-3,3-diphenyl-1-pyrroline (EMDP) of less than 1.0%. 21.A method for detecting EDDP in a sample, comprising the steps of:a)combining the sample with the antibody of claim 15 under conditions thatpermit formation of a stable EDDP-antibody complex; and b) detecting anyEDDP-antibody complex formed in step a).
 22. The method of claim 21,comprising quantitating any EDDP-antibody complex formed in step a). 23.The method of claim 21, which is a competition assay method.
 24. Themethod of claim 21, further comprising the steps of:i) contacting theantibody with a labeled analog of EDDP under conditions that permitformation of a stable analog-antibody complex; and ii) separating anyanalog not forming a complex in step i).
 25. The method of claim 21,which is a homogeneous assay method.
 26. The method of claim 21, whichis a cloned enzyme donor immunoassay method.
 27. The method of claim 21,comprising the steps of:i) combining the sample with:said antibody, anenzyme donor polypeptide conjugate according to the compound of claim 3,an enzyme acceptor polypeptide wherein said enzyme acceptor polypeptidecapable of forming with said enzyme donor polypeptide conjugate anactive enzyme complex in the absence of an antibody to EDDP, and asubstrate capable of being transformed by the active enzyme complex intoa product; and ii) measuring the rate of product formation.
 28. Themethod of claim 27, wherein the active enzyme complex hasβ-galactosidase activity.
 29. A method for generating an antibodyspecific for EDDP, comprising immunizing a mammal or contacting animmunocompetent cell or virus with the compound of claim
 11. 30. Adiagnostic kit for measuring EDDP in a sample, comprising the antibodyof claim 15 in suitable packaging.
 31. A diagnostic kit for measuringEDDP in a sample, comprising the compound of claim 12 in suitablepackaging.
 32. A method for making a diagnostic kit for measuring EDDPin a sample, comprising packaging the antibody of claim
 15. 33. A methodfor making a diagnostic kit for measuring EDDP in a sample, comprisingpackaging the compound of claim
 1. 34. An antibody to the methadonemetabolite EDDP having a crossreactivity with methadone of less than0.5%.
 35. An antibody to the methadone metabolite EDDP having acrossreactivity with methadone of less than 0.1%.